Women have a dramatically lower incidence of heart disease than age-matched men, but this difference is largely erased after menopause. Further investigation has indicated that this is because estrogen has many important functions in normal vascular biology and atheroprotection. However, estrogen treatment also has detrimental side effects, including an increased risk of endometrial and breast cancers, and thromboembolic events resulting from increased production of coagulation factors in the liver. Significantly, despite many years of study, the molecular mechanisms underlying the tissue specific effects of estrogen have yet to be elucidated. The functions of estrogen in the vasculature and other tissues are mediated by the estrogen receptors (ERs) alpha and beta (ER1 & ER2). ERs are transcription factors, that, when bound by an agonist such as 172 estradiol (E2), bind to specific chromatin locations to activate or repress transcription. Our recent RNA-seq and microarray studies have shown that E2 regulates very different sets of genes in mouse liver versus mouse aorta, and also that distinct sets of genes are regulated in mouse aorta over increasing times of E2 treatment. Strikingly, our recent ChIP-seq and bioinformatic studies also indicate that only a small fraction of the genomewide ER1 binding sites in mouse aorta overlap with ER1 binding sites in mouse liver, and that consensus sequences for different transcription factors are enriched in the E2-responsive promoters and ER1 binding sites from each tissue. These preliminary studies support the hypothesis that the tissue-specific and time-dependent responses to E2 are determined by differences, between tissues and over time of E2 exposure, in the patterns of ER1 on chromatin and in the nature of cooperating transcription factors. To test this hypothesis, we will use cutting edge genomewide approaches to correlate and compare temporal changes in ER1 recruitment and gene expression in mouse aorta, uterus and liver. These tissues were chosen because they are excellent models for E2 functions in atheroprotection, cancer, and in a combination of beneficial and harmful gene expression changes related to lipid metabolism and thrombosis. Bioinformatic analysis of these data will identify transcription factors in each tissue that are likely to cooperate with ER1 at its binding sites on chromatin, or act independently of ER1 binding in cis to modulate the transcriptional effects of E2. These studies will be, to our knowledge, the first to compare ER distribution and regulatory functions at different times and in multiple whole tissues. Accordingly, they are likely to offer important new insights into the general mechanisms of gene regulation by estrogen. In addition, these studies will identify and begin to test the functions of candidate factors which may mediate vascular specific atheroprotective transcriptional responses. Importantly, in future studies, the new regulatory models discovered in these studies will be readily testable by combining the genomics approaches developed here with powerful mouse transgenic and pharmacological models of vascular ER function that are in use or being developed in our research group. PUBLIC HEALTH RELEVANCE: There are currently no adequate models to explain the differential effects of estrogen in vascular versus other tissues, and this limits the development of therapeutics that could capitalize on the atheroprotective effects of estrogen. The successful completion of these studies is expected to provide novel insights into the mechanism of time-dependent and tissue specific gene regulation by estrogen, and to identify blood vessel-specific transcription factors that mediate the vasoprotective effects of estrogen. Given that these factors are likely to be regulated by signaling pathways that can be manipulated by a variety of drugs, these studies have the long-term potential to contribute to the development of novel therapies that promote the vascular protective effects of estrogen without its associated deleterious side effects, which include breast and uterine cancers, and deep vein thrombosis.